Researchers tag proteins with fluorescent amino acid

Researchers at The Scripps Research Institute in La Jolla, Calif.,
have developed a way to genetically encode a fluorescent amino acid in a cell. The
result is a fluorescent tag with a lower molecular weight than GFP and one that
can be used to measure the expression of proteins, and of protein folding, binding
or trafficking inside live cells.

According to Scripps researcher Daniel Summerer,
the ability to genetically encode GFP has been extremely useful in molecular biology
because it overcomes many of the limitations of chemical fluorescent probes, such
as yield and selectivity. However, GFP is a large molecule, which means that it
can upset the natural structure of the protein to which it is attached. Moreover,
its size makes relative distance measurements difficult, and it can be attached
only at specific locations on a protein.

The researchers developed a method
for encoding dansylalanine into yeast cells. The dansyl chromophore is a widely
used biochemical probe that is small and highly sensitive to its local molecular
environment and that has an extremely large Stokes shift, Summerer said.

“The size of the dansyl side
chain is only slightly smaller than that of the naturally occurring amino acid tryptophane,
making it likely that it can be accommodated in proteins without significant structural
perturbation,” he said, adding that it is only a single amino acid, whereas
GFP has more than 230 amino acids.

A new technique allows researchers to genetically engineer a yeast
strain, MJY125, to express a fluorescent amino acid. Courtesy of Daniel Summerer.
After successfully encoding the fluorescent
amino acid in yeast cells, the researchers demonstrated its functionality as a probe
of protein unfolding.

They encoded the amino acid at positions
16 and 33 in the human superoxide dismutase protein, which has a β-barrel
fold. In the naturally folded state of the proteins, the fluorescence of the dansyl
moiety in the two proteins increased slightly, and its emission spectra shifted
to the blue when compared with the unbound amino acid. To unfold the protein, the
researchers applied increasing concentrations of guanidinium chloride. They observed
changes in fluorescence intensity and emission wavelength of the fluorescent amino
acid that indicated that the local molecular environment of the two amino acid positions
is different during the unfolding process.

Summerer said that the method incorporates
the benefits of both fluorescent proteins and of small organic dyes. “It uses
a small chromophore, but it can be applied in vivo like fluorescent proteins, making
it interesting for experiments in living organisms,” he said. “However,
it also will facilitate the production of large amounts of fluorescently tagged
proteins with technical ease for in vitro applications such as diagnostics or proteomics.”

The researchers continue to work on
the technology. To date, they have expanded it to mammalian cell lines and are working
on in vivo imaging experiments. They also have added a second fluorophore, coumarin,
into proteins in bacteria. They plan to pursue this work as well with the goal of
developing a large set of fluorophores for a wide variety of applications.